Methods of manufacturing bioactive gels from extracellular matrix material

ABSTRACT

The present invention is directed to methods of manufacturing bioactive gels from ECM material, i.e., gels which retain bioactivity, and can serve as scaffolds for preclinical and clinical tissue engineering and regenerative medicine approaches to tissue reconstruction. The manufacturing methods take advantage of a new recognition that bioactive gels from ECM material can be created by digesting particularized ECM material in an alkaline environment and neutralizing to provide bioactive gels.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to and benefit of U.S. ProvisionalApplication No. 61/702,437, filed Feb. 8, 2013, the entire contents ofwhich are incorporated by reference herein for all purposes.

GOVERNMENT SUPPORT

This invention was supported by grant no. NCC 9-58 from the NationalSpace Biomedical Research Institute through NASA. The Government hascertain rights in the invention.

TECHNICAL FIELD OF THE INVENTION

The present invention is related to methods of manufacturing bioactivegels from extracellular matrix material and their uses for restorationof tissues in a patient.

BACKGROUND

Biologic scaffolds composed of extracellular matrix material (ECM) havebeen used for the repair of variety of tissues including the lowerurinary tract, esophagus, myocardium and musculotendinous tissues, oftenleading to tissue-specific constructive remodeling with minimal or noscar tissue formation.

Although uses of ECM as scaffolds for preclinical and clinical tissueengineering and regenerative medicine approaches to tissuereconstruction are very promising, challenges remain in the process tomanufacture bioactive gels from ECM, which retain their bioactivity.

The methods of manufacturing bioactive gels from ECM described in theprior art require the use of enzymes and are time consuming because theyrequire aggressive purification steps, which may lead to depletion inthe bioactivity of the gels and may present additional regulatorybarriers to marketing.

Thus, a need exists to manufacture bioactive gels from ECM which avoidscumbersome preparation and purification steps yet result in gels thatretain the bioactivity of the original material.

SUMMARY OF THE INVENTION

The present invention generally pertains to improved methods ofmanufacturing bioactive gels from ECM which retain sufficientbioactivity to positively assist in tissue repair. The present inventionutilizes reagents that do not introduce additional regulatory burdensfor market approval or clearance of the gel invention. Thus, theinvention describes methods of manufacturing bioactive gels from an ECMcomprising (a) providing the ECM from one or more of the groupconsisting of but not limited to small intestine submucosa (SIS),urinary bladder submucosa (UBS), urinary bladder matrix (UBM) (includesepithelial basement membrane), porcine dermis (PD), and liver basementmembrane (LBM), (b) particularizing the ECM to a particle size in therange of about 1 μm to about 1000 μm, (c) solubilizing concentrations inthe range of about 0.5 to 11% weight/volume (w/v) of particularizedpowder in sodium hydroxide (NaOH) in the range of 0.1 to 1.0 M forperiods of time ranging from about 1 hour to about 48 hours at 4° C.,(d) neutralizing the solubilized ECM prepared in step (c) withhydrochloric acid (HCl), optionally equimolar relative to NaOH, rangingfrom 0.1 to 1 M to form the gel, and (e) optionally, freezing theneutralized solubilized ECM prepared in step (d), optionally (f)lyophilizing the frozen ECM prepared in step (e), and optionally (f)reconstituting the lyophilized gel in water or saline.

The advantages provided by the methods of manufacturing bioactive gelsin the above manner are that aggressive purification steps, which aredeleterious to bioactivity, tedious to perform or are time consuming,and which increase the regulatory burden (e.g. FDA approval), areavoided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the FGF-2 content (pg/mg) of gels following varioussolubilization conditions in NaOH according to embodiments of theinvention. All gels not marked with a % w/v were done at 7.0% w/v UBM toNaOH. All gels in FIG. 1 were done at 7.0% w/v UBM to NaOH.

FIG. 2 shows the VEGF content (pg/mg) of gels following varioussolubilization conditions in NaOH according to embodiments of theinvention. All gels not marked with a % w/v were done at 7.0% w/v UBM toNaOH. All gels in FIG. 1 were done at 7.0% w/v UBM to NaOH.

DETAIL DESCRIPTION

The present invention is directed to methods of manufacturing bioactivegels from ECM, i.e., gels which retain sufficient bioactivity topositively assist tissue repair by decreasing the time needed forrepair, decreasing scar tissue formation, and improving restoration ofthe injured tissue to its pre-damaged native structure and function ascompared to injured tissues not treated with the bioactive gel accordingto the invention. The gel invention and methods of making describedherein serves as scaffolds for preclinical and clinical tissueengineering and regenerative medicine approaches to tissuereconstruction. Bioactivity in the ECM gel according to the invention isin the range of about 0 to 100%, 25-75%, 10-25%, less than 10%, lessthan 5% or less than 1% of the bioactivity of one or more bioactivemolecules in the native ECM from which the gel was derived. As will bedescribed in detail below, these manufacturing methods take advantage ofa new recognition that bioactive gels from ECM can be created bysolubilizing a particularized ECM in a basic (greater than pH 7)environment, which when neutralized with acid provides bioactive gels.

In accordance with the inventive methods, the ECM may be derived fromlayers of native mammalian tissues including but not limited tosubmucosa, dermis, epithelial basement membrane, or from tissues such asaponeurosis, fascia, tendon, ligament, smooth and skeletal muscle andtreatment site-specific ECM. The native mammalian tissue source may beporcine, bovine, ovine, allogenic, or autogenic, for example. Forexample, the ECM may be SIS (small intestinal submucosa), UBS (urinarybladder submucosa) or UBM (urinary bladder matrix) or liver basementmembrane (LBM) described in U.S. Pat. No. 6,576,265, U.S. Pat. No.6,579,538, U.S. Pat. No. 5,573,784, U.S. Pat. No. 5,554,389, U.S. Pat.No. 4,956,178, and U.S. Pat. No. 4,902,508, each of which areincorporated by reference herein. In one embodiment of the invention,the ECM is derived from a mammalian tissue and comprises bioactivecomponents of the extracellular matrix material that are arranged and inquantities similar to those in the tissue in its native form.

In accordance with the inventive method, the ECM derived from any one ofthe above sources is particularized, i.e., the size of the ECM particlesare in a range of about 1 μm to about 1000 μm. In one embodiment,particularization of the ECM prior to subjecting the ECM to a basicenvironment provides homogeneity to the ECM, i.e., provides a moreuniform composition in comparison to ECM from individual animals,decreasing the impact of inter-donor variability. In another embodiment,the particularization of the ECM facilitates in solubilizing the matrixin a basic environment by increasing surface area to volume ratio.

The particulate ECM product, e.g., particularized ECM, is manufacturedby grinding/milling or otherwise performing a size reduction process toECM typically but not exclusively originally provided in sheet form. Theresulting particulate can be any desired range of density for example inthe range of about 0.1 g/cm³-10 g/cm³, about 0.10.1 g/cm³-1 g/cm³ orabout 1 g/cm³, and particle size for example in the range of about 1micron-1000 microns, about 200-700 microns, about 300-600 microns, orabout 400 microns.

A basic environment is provided by solutions of alkaline compounds.Alkaline compounds which could be used in accordance with the inventionare metal hydroxides which include, but are not limited to, LiOH, NaOH,KOH, RbOH, and CsOH. Alkaline compounds which could be used inaccordance with the invention also include weak bases, such as but notlimited to, ammonia (NH₃), pyridine (C₅H₅N), hydroxylamine (H₂NOH),methylamine (NH₂CH₃) and the like. Alkaline compounds are generally usedat a concentration ranging from 0.1 Molar to 1.0 Molar, althoughconcentrations lower that 0.1 Molar or higher than 1.0 Molar are alsocontemplated in an embodiment of the invention.

Concentrations of particularized ECM to NaOH (w/v) are in the range of0.1% to about 20%, in particular 0.5% to 11%, and more particular, 7%.

The solubilization step at 4° C. (i.e., digestion) of the particularizedECM can extend over a period of time ranging from a few minutes toseveral hours (e.g., 30 minutes to 48 hours) or days (e.g., 3-7 days),30 minutes to 12 hours, 12-24 hours, 24 to 36 hours, 36 to 48 hours, ortwo to seven days. In an embodiment of the invention, it is contemplatedthat the time period required for the digestion step is determined bythe size of the particularized extracellular matrix material and/or theconcentration of the metal hydroxide used for solubilization. Forexample, if the concentration of an alkaline compound, such as NaOH, islow, longer incubation, i.e., longer time period for solubilization maybe required. After the solubilization step in a basic solution, thesolubilized ECM (i.e., the gel form) is neutralized to a neutral pHusing molar concentrations, e.g., equimolar concentrations of an acid ina volume sufficient for the solubilized ECM to reach pH 6.8 to 7.4.Acids, which aid in neutralization of the ECM gel, can be selected fromweak or strong acids. Selectivity of acids for the neutralization stepdepends on the salts which are produced when an acids reacts with thebasic environment during neutralization. The resulting salt should bebiocompatible. For example, in an embodiment of the invention,hydrochloric acid (HCl) is used to neutralize the basic environmentcreated by the base NaOH because the resulting salt (i.e., NaCl) isclinically acceptable.

Following neutralization, optionally, gels can be subjected to variousdwell periods, 1-48 hours, 12-36 hours, or 36 to 48 hours to promoterefolding of denatured bioactive components. Dwell periods are generallyperformed with or without shaking or stirring the gel in a cold room(i.e., at temperature of about 4° C.), alternatively at roomtemperature. Dwell periods could extend beyond 48 hours to a few days,for example, 3-7 days to promote restructuring of the gel.

According to the method of the invention, once the gel is neutralized itmay optionally be subjected to one or more steps of freeze drying cyclesto facilitate the conversion of the neutralized gel to a powder (havinga neutral pH). The powder can be reconstituted into a gel withoutaltering its bioactivity by mixing the powder with a liquid, such aswater, or a buffer solution which maintains the neutral pH of the gel.In addition, preservation of bioactivity of ECMs may be achieved throughlyophilization.

In one embodiment according to the invention, the freeze dried,solubilized ECM is reconstituted using water and two 3 mL syringes. Onesyringe contains the lyophilized gel, the other water, and they aremixed together via a connecter between the two syringes. The mixture isinjected back and forth several times to achieve mixing. Variousconcentrations of ECM in NaOH can be tested for handling properties(i.e. injectability, tackiness, viscosity) to determine their ability tobe applied using the two syringe system. The final consistency of allgels is foam-like, and each one adheres to the surface to which it isapplied while also maintaining consistency, which may be desirable inzero gravity conditions, for example, in space. Accordingly, the gelinvention may be used for tissue repair during space exploration.

In one embodiment, to the solubilized and neutralized ECM gel,particularized ECM is added to increase the viscosity or the bioactivityof the gel. For example, UBM powder in the size range of about 1micron-1000 microns, about 200-700 microns, about 300-600 microns, about100 to 400 microns, about 200 microns or about 400 microns is added to aUBM gel prepared by the above methods to enhance the viscosity or thebioactivity of the gel, that is, the gel has better handling or the gelis made capable of producing a higher concentration of bioactivemolecules, for example, growth factors, such as FGF, e.g., FGF-2, CTGF,or VEGF. ECM powder can be added, prior to, during, or after the gelsare neutralized.

In a particular embodiment, 7.0% UBM to 100 mM NaOH solubilized at 4° C.is used to manufacture the bioactive gel. A dwell period may or may notbe used. UBM powder may be added to the gel to increase viscosity and/orbioactivity.

EXEMPLIFICATIONS

For the following exemplifications, any number of ECM products such asbut not limited to one or more of isolated urinary bladder submucosa,small intestinal submucosa, dermis, for example, could be used. In thefollowing exemplifications, UBM, an ECM isolated from the urinarybladder and having epithelial basement membrane is used as an exemplaryECM. However the invention disclosed herein is not limited to UBM and isapplicable to any isolated ECM.

In an exemplification, gels were created using various concentrations ofparticularized UBM (0.5-11% w/v) solubilized in various concentrationsof NaOH (0.1-1.0 M). UBM was solubilized for various time periods (1-48hours) in its respective concentration of UBM and NaOH at 4° C. In orderto test whether the UBM could restructure after solubilization, gelswere also made using various dwell periods (1-48 hours) followingneutralization.

UBM gels created in the above manner were tested in vitro for bioactivemolecular content. In this study, growth factor (e.g., FGF-2, CTGF,VEGF) content was analyzed. Data for FGF-2 and VEGF content followingsolubilization for each gel structure is shown in FIGS. 1 and 2. Lowerconcentration gels (1-6%) are not shown but produced similar results. Asshown in FIGS. 1 and 2, FGF-2 and VEGF, particularly VEGF levelsincreased in these studies.

In one study it was found that using 7.0% w/v UBM to various range ofNaOH with 24 hours of solubilizing at 4° C. and no dwell period hadsignificantly influenced the FGF-2 and VEGF contents in the gel. FGF-2and VEGF contents were measure by standard ELISA procedures.

What is claimed is: 1-14. (canceled)
 15. A bioactive gel compositioncomprising: an extracellular matrix material (ECM) digested in a basicsolution Wherein the ECM is derived from the extracellular matrix of amammal, said bioactive further comprising a concentration of the ECM toa base (w/v) in the range of about 0.1% to about 20%, wherein said gelis gelled at room temperature.
 16. The composition of claim 15 furthercomprising a neutralizing solution, said neutralizing solution being anacid, said neutralizing solution is added in a volume sufficient tosolubilize the gelled gel and bring the pH to a range between about 6.8to about 7.4.
 17. The composition of claim 15 wherein said gel furthercomprises particularized ECM.
 18. The composition of claim 17 whereinsaid particularized ECM comprises urinary bladder matrix (UBM).
 19. Thecomposition of claim 15 wherein said ECM comprises epithelial basementmembrane.
 20. The composition of claim 15 wherein said base comprisesNaOH.
 21. The composition of claim 16 wherein said composition islyophilized.
 22. composition of claim 21 wherein said lyophilizedcomposition is reconstituted in a solution.
 23. The composition of claim20 wherein said base comprises 0.1-1.0 M NaOH.
 24. The composition ofclaim 15 wherein said base is selected from the group consisting ofLiOH, NaOH, KOH, RbOH, CsOH, NH₃, C₅H₅N, H₂NOH, and NH₂CH₃.
 25. Thecomposition of claim 15 further comprising a bioactive componentselected from the group consisting of VEGF, FGF-2, CTGF, andcombinations thereof.
 26. The composition of claim 15 wherein said ECMcomprises submucosa.
 27. The composition of claim 15 wherein said ECM isselected from the group consisting of submucosa, tunica propria, dermis,liver basement membrane, and combinations thereof.
 28. The compositionof claim 15 wherein the concentration of ECM to NaOH (w/y) is in therange of 0.5% to 11%.
 29. The composition of claim 17 wherein saidparticularized ECM comprises small intestinal submucosa (SIS).
 30. Thecomposition of claim 17 wherein said particularized ECM comprisesurinary bladder submucosa (UBS).
 31. The composition of claim 17 whereinsaid particularized ECM comprises liver basement membrane (LBM).